title: Ethanol decreases cellular protein content and mitochondrial membrane potential of cultured neonatal rat cardiomyocytes: microassays with fluorometric and spectrometric plate readers.
abstract: BACKGROUND
  Ethanol decreases protein synthesis and mitochondrial respiration of the heart. We developed a new time-saving, non-radioactive, small-scale method to determine ethanol-induced alteration in cellular protein content of the cells cultivated in a 96-well plate. To verify the utility of our small-scale method, we used endothelin-1 (ET-1) as a positive control.
  
  
  METHODS
  Neonatal rat cardiomyocytes were cultivated in a 96-well plate and exposed to ethanol (10, 50 and 100 mM) or ET-1 (100 nM). The protein content per cell was determined by measuring both the DNA (Hoechst 33258) and protein amounts of the same well with fluorometric and spectrometric plate readers. We also examined ultrastructure, mitochondrial membrane potential (deltapsim) using JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide), anaerobic glycolysis measured by lactate amount released to the medium, and superoxide production using dihydroethidium.
  
  
  RESULTS
  As expected, ethanol dose- and time-dependently decreased protein content per cell of cardiomyocytes at 24 and 48 h, while ET-1 time-dependently increased it. Ultrastructurally, ethanol decreased rough endoplasmic reticulum at 24 h, whereas ET-1 increased it as well as Golgi apparatus. Among ethanol groups, only 100 mM ethanol affected deltapsim: hyperpolarization just after exposure but slight depolarization at 24 h. ET-1 significantly depolarized deltapsim at 24 h, but the depolarization was within the physiological fluctuation. Ethanol did not change the lactate release at 3 and 24 h. Ethanol did not increase superoxide production at 24 h, but rather 10 mM ethanol significantly decreased it.
  
  
  CONCLUSIONS
  Our small-scale method was able to demonstrate that ethanol and ET-1 induced expected alterations in protein content per cell with dose- and time-dependencies and statistically significant differences compared with the control, thereby validating the practical use of this method. Ultrastructural alterations supported the biochemical results. The ethanol-decreased cellular protein content might partly result from energy shortage attributable to reduced mitochondrial respiration, which was not compensated by anaerobic glycolysis.
authors.affiliation: Biochemistry and Molecular Biology, School of Biomedical, Biomolecular and Chemical Sciences, Faculty of Life and Physical Sciences, The University of Western Australia, 35, Stirling Hwy, Crawley, Western Australia 6009, Australia.
authors.forename: Keiko
authors.initials: K
authors.lastname: Mashimo
authors.forename: Shigeru
authors.initials: S
authors.lastname: Sato
authors.forename: Akiko
authors.initials: A
authors.lastname: Adachi
authors.forename: Peter G
authors.initials: PG
authors.lastname: Arthur
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology*; D005470:Fluorometry / Q000295:instrumentation; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000187:drug effects*; D011506:Proteins / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013057:Spectrum Analysis / Q000295:instrumentation
chemical_list: D019332:Endothelin-1; D011506:Proteins; D000431:Ethanol